Elucidation of Chemical Interactions between Crude Drugs Using Quantitative Thin-Layer Chromatography Analysis.

To elucidate the interactions between crude drugs in Kampo medicines (traditional Japanese medicines), it is important to determine the content of the constituents in a cost-effective and simple manner. In this study, we quantified the constituents in crude drug extracts using thin-layer chromatography (TLC), an inexpensive and simple analytical method, to elucidate the chemical interactions between crude drugs. We focused on five crude drugs, for which quantitative high-performance liquid chromatography (HPLC) methods are stipulated in the Japanese Pharmacopoeia XVIII (JP XVIII) and compared the analytical data of HPLC and TLC, confirming that the TLC results corresponded with the HPLC data and satisfied the criteria of JP XVIII. (Z)-ligustilide, a major constituent in Japanese Angelica Root, for which a method of quantification has not been stipulated in JP XVIII, was also quantitatively analyzed using HPLC and TLC. Furthermore, Japanese Angelica Root was combined with 26 crude drugs to observe the variation in the (Z)-ligustilide content from each combination by TLC. The results revealed that combinations with Phellodendron Bark, Citrus Unshiu Peel, Scutellaria Root, Coptis Rhizome, Gardenia Fruit, and Peony Root increased the (Z)-ligustilide content. Quantifying the constituents in crude drug extracts using the inexpensive and simple TLC method can contribute to elucidating interactions between crude drugs in Kampo medicines, as proposed by the herbal-pair theory.

Synthesis of the Carbohydrate Moiety of Glycoproteins from the Parasite Echinococcus granulosus and Their Antigenicity against Human Sera.

Stereocontrolled syntheses of biotin-labeled oligosaccharide portions containing the carbohydrate moiety of glycoprotein from Echinococcus granulosus have been accomplished. Trisaccharide Galß1-3Galß1-3GalNAcα1-R (A), tetrasaccharide Galα1-4Galß1-3Galß1-3GalNAcα1-R (B), and pentasaccharide Galα1-4Galß1-3Galß1-3Galß1-3GalNAcα1-R (C), (R = biotinylated probe) were synthesized by stepwise condensation and/or block synthesis by the use of 5-(methoxycarbonyl)pentyl 2-azido-4,6-O-benzylidene-2-deoxy-α-d-galactopyranoside as a common glycosyl acceptor. The synthesis of the tetrasaccharide and the pentasaccharide was improved from the viewpoint of reducing the number of synthetic steps and increasing the total yield by changing from stepwise condensation to block synthesis. Moreover, hexasaccharide E, which contains the oligosaccharide sequence which occurs in E. granulosus, was synthesized from trisaccharide D. We examined the antigenicity of these five oligosaccharides by an enzyme-linked immunosorbent assay (ELISA). Although compounds of C-E did not exhibit antigenicity against cystic echinococcosis (CE) patient sera, compounds B, D, and E showed good serodiagnostic potential for alveolar echinococcosis (AE).

Structures and Inhibitory Activities for Interleukin-2 Production of Seasonally Variable Constituents in Flower Parts of Magnolia kobus at Different Growth Stages.

Magnolia Flower is a crude drug used for the treatment of headaches, toothaches, and nasal congestion. Here, we focused on Magnolia kobus, one of the botanical origins of Magnolia Flower, and collected the flower parts at different growth stages to compare chemical compositions and investigate potential inhibitory activities against interleukin-2 (IL-2) production in murine splenic T cells. After determining the structures, we examined the inhibitory effects of the constituents of the bud, the medicinal part of the crude drug, against IL-2 production. We first extracted the flower parts of M. kobus from the bud to fallen bloom stages and analysed the chemical compositions to identify the constituents characteristic to the buds. We found that the inhibitory activity of the buds against IL-2 production was more potent than that of the blooms. We isolated two known compounds, tiliroside (1) and syringin (2), characteristic to the buds from the methanol (MeOH) extract of Magnolia Flower. Moreover, we examined the inhibitory activities of both compounds against IL-2 production and found that tiliroside (1) but not syringin (2), showed strong inhibitory activity against IL-2 production and inhibited its mRNA expression. Thus, our strategy to examine the relationship between chemical compositions and biological activities during plant maturation could not only contribute to the scientific evaluation of medicinal parts of crude drugs but also assist in identifying biologically active constituents that have not yet been reported.

Synthesis of the Non Reducing End Oligosaccharides of Glycosphingolipids from Ascaris suum.

Stereocontrolled syntheses of biotin-labeled oligosaccharide portions containing the non reducing end oligosaccharides of glycosphingolipids from Ascaris suum have been accomplished. Galα1→3GalNAcß1→OR (1), Galß1→3Galα1→3GalNAcß1→OR (2), Galß1→6Galα1→3GalNAcß1→OR (3), Galß1→6(Galß1→3)Galα1→3GalNAcß1→OR (4) and GlcNAcß1→6Galß1→6(Galß1→3)Galα1→3GalNAcß1→OR (5) (R = biotinylated probe) were synthesized by stepwise condensation (1-4) and block synthesis (5) using 5-(methoxycarbonylpentyl) 2-O-benzoyl-3-O-2-napthylmethyl-4,6-O-di-tert-butylsilylene-α-D-galactopyranosyl-(1→3)-4,6-O-benzylidene-2-deoxy-2-phthalimido-ß-D-galactopyranoside (12) as a common precursor. Compound 12 was converted into two kinds of glycosyl acceptors and was condensed with suitable galactosyl donors, respectively.

Synthesis and Antigenicity against Human Sera of a Biotin-Labeled Oligosaccharide Portion of a Glycosphingolipid from the Parasite Echinococcus multilocularis.

Synthesis of a biotinylated analog of the carbohydrate portion of a glycosphingolipid from the parasite Echinococcus multilocularis has been achieved. We synthesized ß-D-Galp-(1→6)-ß-D-Galp-(1→6)-[α-L-Fucp-(1→3)]-ß-D-Galp-(1→R: biotin probe) (1) and compared the antigenicity by an enzyme linked immunosorbent assay (ELISA) with biotinylated trisaccharide α-D-Galp-(1→4)-ß-D-Galp-(1→3)-α-D-Galp-(1→R: biotin probe) (F), which has been shown to have significant antigenicity. Both of the oligosaccharides reacted with sera of alveolar echinococcosis (AE) patients, but showed different reactivity. Among the 60 sera of AE patients, more sera reacted with the linear sequence Galα1→4Galß1→3GalNAcα1→R of oligosaccharide (F) than for branched compound 1. Some sera showed high specificity to one of the compound, indicating that the antibodies in the sera of AE patients differ in their specificity to recognize carbohydrate sequences of glycosphingolipids. Our results demonstrate that both of the biotinylated oligosaccharides 1 and F have good serodiagnostic potential and are complementary to detect infections caused by the parasite Echinococcus multilocularis.

The development of new molecular tools containing a chemically synthesized carbohydrate ligand for the elucidation of carbohydrate roles via photoaffinity labeling: carbohydrate-protein interactions are affected by the structures of the glycosidic bonds and the reducing-end sugar.

Photoaffinity labeling technology is a highly efficient method for cloning carbohydrate-binding proteins. When the carbohydrate probes are synthesized according to conventional methods, however, the reducing terminus of the sugar is opened to provide an acyclic structure. Our continued efforts to solve this problem led to the development of new molecular tools with an oligosaccharide structure that contains a phenyldiazirine group for the elucidation of carbohydrate-protein interactions. We investigated whether carbohydrate-lectin interactions are affected by differences in the glycosidic formation and synthesized three types of molecular tools containing Galp-GlcpNAc disaccharide ligands and a photoreactive group (1, 2, 3). Photoaffinity labeling validated the recognition of the new ligand by different glycosidic bonds. Photoaffinity labeling also demonstrated that both the reducing end sugar and non-reducing end sugar recognized the Erythrina cristagalli agglutinin.

Scutellaria Root extract-induced hepatocytotoxicity can be controlled by regulating its baicalin content.

Scuellaria Root (SR, root of Scutellaria baicalensis), which has potent anti-inflammatory effects, is a component of useful Kampo formulae. Albeit a low frequency, SR induces serious interstitial pneumonia and liver dysfunction. In this study, to control the adverse effects of SR, we investigated the causal constituent responsible for its hepatocytotoxicity and aimed to develop a method to control it. As a result, we revealed that the hepatocytotoxicity of SR was correlated with its baicalin content, a major constituent in SR. It was confirmed by preparing a baicalin-free SR extract, which exhibited reduced hepatocytotoxicity. The addition of baicalin to the baicalin-free SR extract restored the hepatocytotoxicity, indicating that the hepatocytotoxicity of SR is dependent on its baicalin content. Thus, SR extract-induced hepatocytotoxicity can be controlled by regulating its baicalin content.

Further structural characterization of the Echinococcus granulosus laminated layer carbohydrates: the blood-antigen P1-motif gives rise to branches at different points of the O-glycan chains.

The glycobiology of the cestodes, a class of parasitic flatworms, is still largely unexplored. An important cestode species is Echinococcus granulosus, the tissue-dwelling larval stage of which causes hydatid disease. The E. granulosus larva is protected from the host by a massive mucin-based extracellular matrix termed laminated layer (LL). We previously reported ( Díaz et al. 2009. Biochemistry 48:11678-11691) the molecular structure of the most abundant LL O-glycans, comprising up to six monosaccharide residues. These are based on Cores 1 and 2, in cases elongated by a chain of Galpß1-3 residues, which can be capped by Galpα1-4. In addition, the Core 2 GlcNAcp residue can be decorated with the Galpα1-4Galpß1-4 disaccharide. Larger glycans also detected contained additional HexNAc residues that could not be explained by the structural repertoire described above. In this work, we elucidate, by mass spectrometry (MS) and nuclear magnetic resonance (NMR), six additional glycans from the E. granulosus LL between six and eight residues in size. Their structures are related to those already described but in cases bear GlcNAcpß1-6 or Galpα1-4Galpß1-4GlcNAcpß1-6 as ramifications on the core Galpß1-3 residue. We also obtained evidence that noncore Galpß1-3 residues can be similarly ramified. Thus, the new motif together with the previous information may explain all the glycan compositions detected in the LL by MS. In addition, we show that the anti-Echinococcus monoclonal antibody E492 (Parasite Immunol 21:141, 1999) recognizes Galpα1-4Galpß1-4GlcNAcp (the blood P(1)-antigen motif). This explains the antibody's reactivity with a range of Echinococcus tissues, as the P(1)-motif is also carried on non-LL N-glycans and glycolipids from this genus.

Sulfatides inhibit adhesion, migration, and invasion of murine melanoma B16F10 cell line in vitro.

Endogenous sulfatide, such as 3-sulfated galactosylceramide (3-sulfatide) has been reported to be involved in neuronal development and regulation of tumor cell metastasis. Recently, a new 6-sulfated glucosylceramide (6-sulfatide) has been isolated from the ascidian, Ciona intestinalis. To determine the antitumor function of the new sulfatide, we examined the effects of synthetic 6-sulfatide and 3-sulfatide on the metastatic features of a murine melanoma cell line, B16F10. Both sulfatides significantly inhibited the adhesion of melanoma cells onto fibronectin-coated tissue plates and, the motility and invasion of the cells, with 6-sulfatide showing stronger inhibitory activities. In addition, both sulfatides inhibited α(5)-, and ß(1)- but not α(v)- or ß(3)-integrin expression. Furthermore, these sulfatides inhibited the activation of focal adhesion kinase, Akt, and extracellular signal-regulated kinase signaling pathways, which are thought to be important for cell migration and invasion. Therefore, these sulfatides may serve as promising drug candidates for the treatment of cancer metastasis.

Galα1-4Galß1-3GalNAc is the dominant epitope of Em2 antigen, the mucin-type glycoprotein from Echinococcus multilocularis.

The larval stage of Echinococcus multilocularis causes alveolar echinococcosis in human. In serodiagnosis of alveolar echinococcosis, specific reactions have been noted not only against protein antigens but also carbohydrates. With regard to protein antigens, the recent development of recombinant antigens has contributed to an improvement in serodiagnostic examination. On the contrary, the preparation of carbohydrate antigen still depends on extraction from crude antigens, and isolation is usually accompanied with difficulty; consequently, it is rare to examine individual antigenicity of carbohydrates. However, parasitic helminths express various antigenic carbohydrates. In the case of Echinococcus granulosus, antigenic glycoproteins of the laminated layer have been reported. Furthermore, the laminated layer of E. multilocularis contains Em2 antigen which is a famous mucin-type glycoprotein and which seems to play an important role in metacestode survival mechanisms within the immunologically reacting host; nevertheless, the anomeric configurations and the individual antigenicity of Em2 O-glycans have not been confirmed so far. Under these circumstances, we introduced a chemical synthesis to get pure oligosaccharides in order to assess diagnostic performance. In our previous study, 11 oligosaccharides have already been prepared by stereocontrolled syntheses. Among them, three synthetic oligosaccharides showed antigenicity. Our aim is to investigate correct sequence and serodiagnostic potential of the dominant epitope of Em2. This study provided important diagnostic information: (1) the trisaccharide Galα1-4Galß1-3GalNAc sequence is the dominant epitope of Em2 (sensitivity 95.0 %), (2) Trematoda expresses carbohydrates with the similar trisaccharide sequence, and (3) the terminal Galα1-4Gal sequence is a candidate for the widely common epitope that accounts for the cross-reaction.

Synthesis, antigenicity against human sera and structure-activity relationships of carbohydrate moieties from Toxocara larvae and their analogues.

Stereocontrolled syntheses of biotin-labeled oligosaccharide portions containing the Galß1-3GalNAc core of the TES-glycoprotein antigen obtained from larvae of the parasite Toxocara and their analogues have been accomplished. Trisaccharides Fuc2Meα1-2Gal4Meß1-3GalNAcα1-OR (A), Fucα1-2Gal4Meß1-3GalNAcα1-OR (B), Fuc2Meα1-2Galß1-3GalNAcα1-OR (C), Fucα1-2Galß1-3GalNAcα1-OR (D) and a disaccharide Fuc2Meα1-2Gal4Meß1-OR (E) (R = biotinylated probe) were synthesized by block synthesis using 5-(methoxycarbonyl)pentyl-2,3,4,6-tetra-O-acetyl-ß-D-galactopyranosyl-(1-->3)-2-azide-4-O-benzyl-2-deoxy-α-D-galactopyranoside as a common glycosyl acceptor. We examined the antigenicity of these five oligosaccharides by enzyme linked immunosorbent assay (ELISA). Our results demonstrate that the O-methyl groups in these oligosaccharides are important for their antigenicity and the biotinylated oligosaccharides A, B, C and E have high serodiagnostic potential to detect infections caused by Toxocara larvae.

Repeated psychological stress, chronic vicarious social defeat stress, evokes irritable bowel syndrome-like symptoms in mice.

Increasing evidence has demonstrated that emotional states and intestinal conditions are inter-connected in so-called "brain-gut interactions." Indeed, many psychiatric disorders are accompanied by gastrointestinal symptoms, such as the irritable bowel syndrome (IBS). However, the functional connection remains elusive, partly because there are few useful experimental animal models. Here, we focused on a highly validated animal model of stress-induced psychiatric disorders, such as depression, known as the chronic vicarious social defeat stress (cVSDS) model mice, which we prepared using exposure to repeated psychological stress, thereafter examining their intestinal conditions. In the charcoal meal test and the capsaicin-induced hyperalgesia test, cVSDS model mice showed a significantly higher intestinal transit ratio and increased visceral pain-related behaviors, respectively. These changes persisted over one month after the stress session. On the other hand, the pathological evaluations of the histological and inflammatory scores of naive and cVSDS model mice did not differ. Furthermore, keishikashakuyakuto-a kampo medicine clinically used for the treatment of IBS-normalized the intestinal motility change in cVSDS model mice. Our results indicate that cVSDS model mice present IBS-like symptoms such as chronic intestinal peristaltic changes and abdominal hyperalgesia without organic lesion. We therefore propose the cVSDS paradigm as a novel animal model of IBS with wide validity, elucidating the correlation between depressive states and intestinal abnormalities.

The effect of structural differences in the reducing terminus of sugars on the binding affinity of carbohydrates and proteins analyzed using photoaffinity labeling.

Because carbohydrates and proteins bind with such low affinity, the nature of their interactions is not clear. Photoaffinity labeling with diazirin groups is useful for elucidating the roles of carbohydrates in these binding processes. However, when carbohydrate probes are synthesized according to this conventional method, the reducing terminus of the sugar is opened to provide an acyclic structure. Because greater elucidation of carbohydrate-protein interactions requires a closed-ring carbohydrate in addition to the photoreactive group, we synthesized new molecular tools. The carbohydrate ligands were synthesized in three steps (glycosylation with allyl alcohol, deprotection, and ozonolysis). Specific binding proteins for carbohydrate ligands were obtained by photoaffinity labeling. Closed ring-type carbohydrate ligands, in which the reducing sugar is closed, bound to lectins more strongly than open ring-type sugars. Carbohydrate to protein binding was observed using AFM.

Synthesis, inhibitory effects on nitric oxide and structure-activity relationships of a glycosphingolipid from the marine sponge Aplysinella rhax and its analogues.

The novel glycosphingolipid, ß-D-GalNAcp(1-->4)[α-D-Fucp(1-->3)]-ß-D-GlcNAcp(1-->)Cer (A), isolated from the marine sponge Aplysinella rhax has a unique structure, with D-fucose and N-acetyl-D-galactosamine moieties attached to a reducing-end N-acetyl-D-glucosamine through an α1-->3 and ß1-->4 linkage, respectively. We synthesized glycolipid 1 and some non-natural di- and trisaccharide analogues 2-6 containing a D-fucose residue. Among these compounds, the natural type showed the most potent nitric oxide (NO) production inhibitory activity against LPS-induced J774.1 cells. Our results indicate that both the presence of a D-Fucα1-3GlcNAc-linkage and the ceramide aglycon portion are crucial for optimal NO inhibition.

Changes in the extracted amounts and seasonally variable constituents of Diospyros kaki at different growth stages.

Persimmon Calyx is a crude drug derived from the persistent calyx of mature fruit of Diospyros kaki Thunberg (Ebenaceae) and is used for the treatment of intractable hiccups. Although there are several reports on the isolation of constituents from Persimmon Calyx, its active constituents have not been elucidated. In this study, by focusing on the medicinal part of Persimmon Calyx, calyx on mature fruit of D. kaki, we examined the changes in the extraction amounts of 3 cultivars of D. kaki ('Hiratanenashi', 'Jiro', and 'Tonewase') to identify and quantify seasonally variable constituents during the maturation process by analysing their chemical compositions. We found that the extraction weight of the calyx, fruit of persimmons, and total tannin content in calyxes were significantly increased during maturation. Lupeol (1), betulinic acid (2), pomolic acid (3), ursolic acid (4), ß-sitosterol (5), rotungenic acid (6), barbinervic acid (7), catechin (8), gallocatechin (9), and sucrose (10) were identified in the calyx of D. kaki. Compounds 1, 6, and 7 were isolated from Persimmon Calyx for the first time. Moreover, the isolated compounds (1-7) and their analogue (oleanolic acid) were quantitatively analysed, and the results showed that the amounts of 4 and oleanolic acid were reduced during maturation, whereas that of 2, 3, 6, and 7 were increased.

Synthetic studies on glycosphingolipids from protostomia phyla: synthesis of glycosphingolipids from the parasite Schistosoma mansoni.

Synthetic access to three neutral glycosphingolipids from the parasite Schistosoma mansoni adult worm has been achieved. These structures differ significantly from those of other parasites and exhibit a unique structural motif termed "schisto-core" consisting of GalNAcbeta1-->4Glcbeta1-->sequence. We have synthesized glycosphingolipids, beta-D-GalNAcp-(1-->4)-beta-D-Glcp-(1-->1)Cer (1), beta-D-GlcNAcp-(1-->3)-beta-D-GalNAcp-(1-->4)-beta-D-Glcp-(1-->1)Cer (2) and beta-D-Galp-(1-->4)-beta-D-GlcNAcp-(1-->3)-beta-D-GalNAcp-(1-->4)-beta-D-Glcp-(1-->1)Cer (3).

Syntheses of glycoclusters containing a phosphocholine residue related to a glycosphingolipid from the earthworm Pheretima hilgendorfi.

Three types of glycoclusters related to an amphoteric glycosphingolipid found in the earthworm Pheretima hilgendorfi were synthesized. The glycoclusters were prepared from a common precursor and a simple approach for the rational design of a glycocluster was developed.

Structural characterization of glycosylinositolphospholipids with a blood group type B sugar unit from the edible mushroom, Hypsizygus marmoreus.

Edible fungi, mushrooms, are a popular food in Japan and over 15 cultured mushroom species are available at the food markets. Recently, constituents or ingredients of edible mushrooms have drawn attention because possibilities have been seen for their medical usage. Mycoglycolipids (basidiolipids) of higher mushrooms have been characterized as glycosylinositolphosphoceramides, having a common core structure of Manalpha1-2Ins1-[PO(4)]-Cer and extensions of Man, Gal, and/or Fuc sugar moieties. Seven mycoglycolipids were purified from the edible mushroom Hypsizygus marmoreus by successive column chromatography on ion exchange Sephadex (DEAE-Sephadex) and silicic acid (Iatrobeads). Their structures were characterized to be Ins1-[PO(4)]-Cer (AGL0), Manalpha1-2Ins1-[PO(4)]-Cer (AGL1), Galbeta1-6Manalpha1-2Ins1-[PO(4)]-Cer (AGL2), Fucalpha1- 2Galbeta1-6Manalpha1-2Ins1-[PO(4)]-Cer (AGL3), Galalpha1-3(Fucalpha1-2)Galbeta1-6Manalpha1-2Ins1-[PO(4)]-Cer (AGL4), Galalpha1-2Galalpha1-3(Fucalpha1-2)Galbeta1-6Manalpha1-2Ins1-[PO(4)]-Cer (AGL5), and Galalpha1-2Galalpha1-2Galalpha1-3(Fucalpha1-2)Galbeta1-6Manalpha1-2Ins1-[PO(4)]-Cer (AGL6) by sugar compositional analysis, methylation analysis, periodate oxidation, partial acid hydrolysis, enzymatic hydrolysis, immunochemical analysis, gas-liquid chromatography (GC), gas chromatography-mass spectrometry (GC-MS), matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), and (1)H-nuclear magnetic resonance spectroscopy (NMR). Ceramide constituents of their mycoglycolipids were composed of phytosphingosine as the sole sphingoid, and mainly 2-hydroxy C22:0 and C24:0 acids as the fatty acids. By immunochemical detection, the terminal structure of AGL4, Galalpha1-3(Fucalpha1-2)Galbeta-, was shown to have blood group type B activity. Galalpha1-2 and its repeating sequence in AGL5 and AGL6 are novel structures on the nonreducing sugar end in mycoglycolipids. These two mycoglycolipids in H. marmoreus distinguish it from other basidiomycetes.

Synthesis of neutral glycosphingolipids from Zygomycetes.

Novel neutral glycosphingolipids (NGSLs) containing Gal-alpha1-->6Gal, previously found in the Zygomycetes species Mucor hiemalis, were synthesized. The structures of these compounds are different from those of other fungal GSLs, and they are expected to be involved in host-parasite interactions. A key step in their synthesis is direct 1,2-cis alpha-selective galactosylation of 4,6-diol tri- and tetrasaccharide acceptors with a galactosyl donor in the presence of N-iodosuccinimide (NIS)/trifluoromethanesulfonic acid (TfOH). The fully protected glycosides were deprotected to give the two target glycosphingolipids.

Synthetic studies on glycosphingolipids from Protostomia phyla: syntheses and biological activities of amphoteric glycolipids containing a phosphocholine residue from the earthworm Pheretima hilgendorfi.

Two types of amphoteric glycosphingolipid found in the earthworm Pheretima hilgendorfi, PC(-->6)-beta-d-Galp-(1-->6)-beta-d-Galp-(1-->1)Cer (1) and PC(-->6)-beta-d-Galp-(1-->6)-beta-d-Galp-(1-->6)-beta-d-Galp-(1-->1)Cer (2), and their derivatives (4, 5) were synthesized. These were examined for their ability to enhance production of interleukin-8 (IL-8), a potent inflammatory cytokine involved in neutrophil chemotaxis, in a TNFalpha-stimulated granulocytic HL-60 cells. Compounds 1 and 2 were found to be potent enhancers of IL-8 production.